SECURE (QLK5-CT-2002-01813) SECURE QLK5-CT-2002-01813.

SECURE (QLK5-CT-2002-01813)SECURE (QLK5-CT-2002-01813)

SECURESECURE

QLK5-CT-2002-01813QLK5-CT-2002-01813


SECURESECURE

StEm Canker of oilseed rape: molecular StEm Canker of oilseed rape: molecular tools and mathematical modelling to tools and mathematical modelling to deploy dUrable REsistance.deploy dUrable REsistance.


SECURESECURE

What is Oilseed rape?What is Oilseed rape?

What is stem canker?What is stem canker?

Current methods of control?Current methods of control?

The need for durable resistanceThe need for durable resistance

Project aims?Project aims?

Who benefits from SECURE?Who benefits from SECURE?


What is oilseed rape (OSR)?What is oilseed rape (OSR)?

Agricultural crop grown Agricultural crop grown throughout most of Europe, SE throughout most of Europe, SE and W Australia and in Canada and W Australia and in Canada and the USA.and the USA.

OSR typically grown in Europe is OSR typically grown in Europe is a “Winter” type - sown in autumn a “Winter” type - sown in autumn and harvested the following and harvested the following summer.summer.

Crop is grown primarily for seed Crop is grown primarily for seed production – seeds are crushed production – seeds are crushed to produce oil (edible, industrial)to produce oil (edible, industrial)


What is “stem canker”?What is “stem canker”?

Globally important Globally important disease of oilseed disease of oilseed raperape

Caused by fungus Caused by fungus called called Leptosphaeria Leptosphaeria maculansmaculans

Infects in autumnInfects in autumn

Major damage at Major damage at harvestharvest

Basal stem canker at end of the growing season


What is “stem canker”?What is “stem canker”?

1. Airborne spores land on young crop in autumn

2. Spores infect to cause leaf lesions

3. Fungus grows down through leaf to…

4. infect the base of the stem

5. Cankers grow during late spring/early summer and can “girdle” stem


Current methods of controlCurrent methods of control

Fungicide (Chemical control)Fungicide (Chemical control) Environmentally unfriendlyEnvironmentally unfriendly Expensive for the farmer (time/money)Expensive for the farmer (time/money)

Cultivation techniquesCultivation techniques Limited in usefulnessLimited in usefulness

Host resistance?Host resistance?

SECURE aimed to increase our understanding of host resistance and the best way to use this resource.


What is host resistance?What is host resistance?

Like us, plants and fungi have genesLike us, plants and fungi have genes Genes control all processes of living thingsGenes control all processes of living things

Some of these genes (resistance genes) Some of these genes (resistance genes) help fight attack by diseases – fungi, help fight attack by diseases – fungi, viruses, bacteria.viruses, bacteria.

However, diseases also have genes However, diseases also have genes (avirulence genes) which correspond and (avirulence genes) which correspond and interact with those in the plantinteract with those in the plant


What is host resistance? - continuedWhat is host resistance? - continued

Interaction between specific corresponding Interaction between specific corresponding resistance and avirulence genes, good for resistance and avirulence genes, good for the plant:the plant: Plant recognises presence of fungusPlant recognises presence of fungus Resistance mechanisms prevent infectionResistance mechanisms prevent infection

However, in the “war” of the genes, However, in the “war” of the genes, sometimes the plant resistance genes are sometimes the plant resistance genes are overcome!overcome!


What is host resistance? - continuedWhat is host resistance? - continued

The fungus mutates to loose the function of The fungus mutates to loose the function of the avirulence genethe avirulence gene No interaction with the plant resistance geneNo interaction with the plant resistance gene The plant no longer recognises the presence The plant no longer recognises the presence

of the fungusof the fungus No resistance responseNo resistance response Infection of the plant!!Infection of the plant!!


The need for durable resistanceThe need for durable resistance

Limited number of plant resistance genes Limited number of plant resistance genes can be used in breeding programmescan be used in breeding programmes

Most are overcome in 3-5 yearsMost are overcome in 3-5 years

However, some resistance genes remain However, some resistance genes remain effective for 5-10 years – why?effective for 5-10 years – why?

““Durable resistance”Durable resistance”


The need for durable resistance –The need for durable resistance –cont’dcont’d

Better understanding of interactions allows Better understanding of interactions allows “smart” use of available resistance“smart” use of available resistance

Allows development of breeding strategies to Allows development of breeding strategies to maximisemaximise durability of resistance. durability of resistance.

Oilseed rape flowers in springtime


Aims of SECUREAims of SECURE

Develop a mechanistic model of the life cycle of Develop a mechanistic model of the life cycle of Leptosphaeria maculansLeptosphaeria maculans (pathogen) (pathogen)

Study molecular mechanisms which generate Study molecular mechanisms which generate virulent isolates of the pathogenvirulent isolates of the pathogen

Analyse effects of genetic background and Analyse effects of genetic background and environment on resistance durabilityenvironment on resistance durability

Construct models and disseminate strategies for Construct models and disseminate strategies for deployment of durable resistancedeployment of durable resistance


Who benefits from SECUREWho benefits from SECURE

People within the European UnionPeople within the European Union Reduced pesticide inputReduced pesticide input Reduced environmental impactReduced environmental impact

Global agricultural industryGlobal agricultural industry Breeding of improved types of oilseed rapeBreeding of improved types of oilseed rape Sustainable oilseed rape productionSustainable oilseed rape production

Global scientific communityGlobal scientific community Increased knowledgeIncreased knowledge Modelling of novel systemModelling of novel system


Visit the websiteVisit the websitewww.secure.rothamsted.ac.uk/www.secure.rothamsted.ac.uk/


Methods developed during Methods developed during SECURESECURE

Ascospore shower inoculation protocolAscospore shower inoculation protocol


Methods developed during Methods developed during SECURESECURE

Green-Fluorescent Protein expressing Green-Fluorescent Protein expressing isolates of isolates of L. maculansL. maculans developed developed

a. Stem of oilseed rape cv. Darmor viewed under white light with visible necrosis at leaf scars of inoculated leaves.

b. The same stem viewed under fluorescent light, showing hyphae of GFP-expressing transformed L. maculans isolate ME24 indicating initial infection of stem through the petiole


Results from Workpackage 1Results from Workpackage 1Development of a life cycle model for Development of a life cycle model for

Leptosphaeria maculansLeptosphaeria maculansDeveloped during the first two years Developed during the first two years (2003-04)(2003-04)

Stepwise modelStepwise model

Validated with data collected during Validated with data collected during SECURESECURE

Published in: Evans et al. (2007). Published in: Evans et al. (2007). Development and validation of a Development and validation of a model to describe the progress phoma model to describe the progress phoma stem canker epidemics in England and stem canker epidemics in England and France. Proceedings of the 12France. Proceedings of the 12thth International Rapeseed Congress, International Rapeseed Congress, Wuhan, China, 26-30 March 2007, 4: Wuhan, China, 26-30 March 2007, 4: 161-164.161-164.

Ascopores

Latent period

Petiole

Canker developmentPhoma spot visible Canker

severity


Results from Workpackage 2Results from Workpackage 2Effects of pathogen variation at Avr loci on Effects of pathogen variation at Avr loci on

durability of resistancedurability of resistanceCloning and functional analysis studies done on Cloning and functional analysis studies done on effector genes effector genes AvrLm1AvrLm1 and and AvrLm6AvrLm6

Three publications to date with more to come:Three publications to date with more to come: Fudal I, Ross S, Gout L, Blaise F, Kuhn ML, Eckert MR, Cattolico L, Bernard-Fudal I, Ross S, Gout L, Blaise F, Kuhn ML, Eckert MR, Cattolico L, Bernard-

Samain S, Balesdent MH, Rouxel T (2007) Heterochromatin-like regions as Samain S, Balesdent MH, Rouxel T (2007) Heterochromatin-like regions as ecological niches for avirulence genes in the ecological niches for avirulence genes in the Leptosphaeria maculansLeptosphaeria maculans genome: genome: Map-based cloning of Map-based cloning of AvrLm6AvrLm6. Molecular Plant-Microbe Interactions 20:459-470. Molecular Plant-Microbe Interactions 20:459-470

Gout L, Fudal I, Kuhn ML, Blaise F, Eckert M, Cattolico L, Balesdent MH, Rouxel Gout L, Fudal I, Kuhn ML, Blaise F, Eckert M, Cattolico L, Balesdent MH, Rouxel T (2006) Lost in the middle of nowhere: the T (2006) Lost in the middle of nowhere: the AvrLm1AvrLm1 avirulence gene of the avirulence gene of the Dothideomycete Dothideomycete Leptosphaeria maculansLeptosphaeria maculans. Molecular Microbiology 60:67-80. Molecular Microbiology 60:67-80

Gout L, Kuhn ML, Vincenot L, Bernard-Samain S, Cattolico L, Barbetti M, Moreno-Gout L, Kuhn ML, Vincenot L, Bernard-Samain S, Cattolico L, Barbetti M, Moreno-Rico O, Balesdent MH, Rouxel T (2007) Genome structure impacts molecular Rico O, Balesdent MH, Rouxel T (2007) Genome structure impacts molecular evolution at the evolution at the AvrLm1AvrLm1 avirulence locus of the plant pathogen avirulence locus of the plant pathogen Leptosphaeria Leptosphaeria maculansmaculans. Environmental Microbiology 9:2978-2992. Environmental Microbiology 9:2978-2992


Results from Workpackage 2 continuedResults from Workpackage 2 continuedEffects of pathogen variation at Avr loci on Effects of pathogen variation at Avr loci on

durability of resistancedurability of resistanceFitness studies using Near Isogenic Isolates (NIIs) for Fitness studies using Near Isogenic Isolates (NIIs) for AvrLm1AvrLm1//avrLm1avrLm1, , AvrLm4AvrLm4//avrLm4avrLm4, , AvrLm6AvrLm6//avrLm6avrLm6

Ascospore shower inoculation method usedAscospore shower inoculation method used

Results suggested a fitness cost was associated with Results suggested a fitness cost was associated with evolution from avirulence to virulence at both evolution from avirulence to virulence at both AvrLm1AvrLm1 and and AvrLm4 AvrLm4 loci. loci.

There was a difference in the strength of the effectThere was a difference in the strength of the effect

Results for Results for AvrLm4 AvrLm4 work published in:work published in: Huang Y-J, Li Z-Q, Evans N, Rouxel T, Fitt BDL, Balesdent M-H Huang Y-J, Li Z-Q, Evans N, Rouxel T, Fitt BDL, Balesdent M-H

(2006). (2006). Fitness cost associated with loss of the AvrLm4 avirulence Fitness cost associated with loss of the AvrLm4 avirulence function in Leptosphaeria maculans (phoma stem canker of function in Leptosphaeria maculans (phoma stem canker of oilseed rape). European Journal of Plant Pathology, oilseed rape). European Journal of Plant Pathology, 114: 77-89114: 77-89


Results from Workpackage 3Results from Workpackage 3

Race structure of Race structure of Leptosphaeria maculansLeptosphaeria maculans population characterised across Europepopulation characterised across Europe

Stachowiak et al., 2006. Frequency of Avirulence Alleles in Field Populations of Leptosphaeria maculans in Europe. European Journal of Plant Pathology 114: 67-75.


Results from Workpackage 3 continuedResults from Workpackage 3 continued

Multi-site evaluation of resistance (2002-2005):Multi-site evaluation of resistance (2002-2005):



Multi-site evaluation of resistance (2002-2005):Multi-site evaluation of resistance (2002-2005):Cultivar Phom)

stem canker%inc

Phoma stem

cankerG2

index

Phoma stem lesions

%inc

Phoma stem lesion

G2 index

Phoma leaf lesions

%inc

Phoma leaf lesion% area

Apex 64.6 2.94 68.7 1.95 64.5 2.09Darmor 59.5 2.13 52.6 1.34 60.0 2.02Darmor-MX 23.8 0.69 50.8 1.45 7.3 0.16

Escort 62.3 2.93 67.5 1.86 58.5 1.60Eurol 73.4 3.54 69.6 2.61 60.9 1.70Eurol-MX 31.2 1.16 80.2 3.69 9.6 0.19Falcon 63.3 2.90 66.3 2.16 53.0 1.44Samourai 78.0 4.06 74.2 2.70 63.2 1.95

Shogun 80.3 4.56 48.9 1.48 68.4 2.22Capitol 77.7 3.49 87.3 3.95* 63.0 1.50**Sed (average) 2.08 0.147 3.81 0.195 2.72 0.218

F test <0.001 <0.001 <0.001 <0.001 <0.001 <0.001

*2005 data only **2003 and 2005 data only



Darmor MX (containg the Rlm6 resistance gene used to investigate Darmor MX (containg the Rlm6 resistance gene used to investigate effect of temperature and leaf wetness on infection.effect of temperature and leaf wetness on infection.

Rlm6Rlm6 resistance found to be temperature sensitive resistance found to be temperature sensitive

Results published in: Results published in: Huang Y-J, Evans N, Li Z-Q, Eckert M, Chevre Huang Y-J, Evans N, Li Z-Q, Eckert M, Chevre A-M, Renard M, Fitt BDL (2006). Temperature and leaf wetness A-M, Renard M, Fitt BDL (2006). Temperature and leaf wetness duration affect phenotypic expression of Rlm6-mediated resistance duration affect phenotypic expression of Rlm6-mediated resistance to Leptosphaeria maculans in Brassica napus. to Leptosphaeria maculans in Brassica napus. New New PhytologistPhytologist, , 170170, 129-141 , 129-141

Symptoms on the leaves of Brassica napus Darmor (lacking Rlm6) and DarmorMX (carrying Rlm6) inoculated with ascospores (without wounding, a,b,c,d,e; with 48 h wetness) or conidia (after wounding, f,g; with 72 h wetness) of Leptosphaeria maculans carrying the effector gene AvrLm6. (a) Large grey lesions on Darmor 11 d after inoculation at 15°C; (b) no visible symptoms on DarmorMX 11 d after inoculation at 15°C; (c) small dark spots (black arrows) and green islands (white arrows) on DarmorMX 18 d after inoculation at 15°C; (d) small dark spots (arrows) on DarmorMX 11 d after inoculation at 20°C; (e) large grey lesions on DarmorMX 16 d after inoculation at 25°C; (f) large grey lesions on Darmor 16 d after inoculation at 25°C; (g) large grey lesions on DarmorMX 16 d after inoculation at 25°C. Bar, 5 mm



GFP (Green Fluorescent Protein) – expressing isolates GFP (Green Fluorescent Protein) – expressing isolates of of L. maculansL. maculans developed developed

GFP – expressing isolates used toGFP – expressing isolates used to

These results were published in the following peer-These results were published in the following peer-reviewed publication: Huang reviewed publication: Huang et alet al., 2009. Quantitative ., 2009. Quantitative resistance to symptomless growth of resistance to symptomless growth of Leptosphaeria Leptosphaeria maculans maculans (phoma stem canker) in (phoma stem canker) in Brassica napusBrassica napus (oilseed rape). (oilseed rape). Plant PathologyPlant Pathology, , 5858, 314-323., 314-323.

a) Stem of oilseed rape cv. Darmor viewed under white light with visible necrosis at leaf scars of inoculated leaves. b) the same stem viewed under fluorescent light, showing hyphae of GFP-expressing transformed L. maculansisolate ME24 indicating initial infection of stem through the petiole.


Results from Workpackage 4Results from Workpackage 4

Modelling investigated the merits of Modelling investigated the merits of pyramiding vs. sequential deployment of pyramiding vs. sequential deployment of resistance genes?resistance genes?


Pyramiding vs. sequential Pyramiding vs. sequential deployment of resistance genes?deployment of resistance genes?

Conclusions:Conclusions:

Fitness cost of virulence high – Pyramid Fitness cost of virulence high – Pyramid resistance genesresistance genes

Fitness cost of virulence low – Sequential Fitness cost of virulence low – Sequential deployment more beneficialdeployment more beneficial


SECURESECURE

For more details on the SECURE project, For more details on the SECURE project, please see the website:please see the website:

www.secure.rothamsted.ac.ukwww.secure.rothamsted.ac.uk


SECURE consortiumSECURE consortium

SECURE (QLK5-CT-2002-01813) SECURE QLK5-CT-2002-01813.

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